CRISPR/Cas9-Based Editing of KLRC1 Enhances the Efficacy of Primary CD33-Targeting CAR-NK Cells Against Acute Myeloid Leukemia

Abstract Text: Chimeric antigen receptor (CAR) natural killer (NK) cells show strong antileukemic activity against acute myeloid leukemia (AML) in vivo. However, NK cell-mediated tumor killing is often impaired by tumor-mediated immune cell inactivation. Acute Myeloid Leukemia (AML) still represents a severe disease with limited therapeutic options, especially for elderly patients. Recently, we reported on the successful generation of primary CD33-targeting CAR(CAR33)-NK cells, which are highly effective against AML in vitro as well as in AML-xenograft mouse models (Albinger et al. BCJ 2022). Yet, CAR-NK cell function can be impaired by high levels of the inhibitory immune checkpoint receptor NKG2A (natural killer group 2A) expressed on NK cells (Bexte et al. Oncoimmunology 2022). Here, we describe a novel strategy to overcome CAR-NK cell inhibition mediated by the immune checkpoint NKG2A, which interacts with HLA-E expressed on AML blasts. We generated AML-targeted CD33-CAR (CAR33)-NK cells combined with CRISPR/Cas9-based gene editing of the NKG2A-encoding KLRC1 gene. Single-cell multi-omics analyses revealed a high proportion of activated cells in CAR33-NK and CAR33-KLRC1ko-NK pools, which was preserved following exposure to AML cells, and which correlated with improved antileukemic activity against AML cell lines and primary blasts in vitro and in vivo. We conclude that dual modified NK cells have the potential not only to bypass the suppressive effect of AML but also of a broad range of other cancer entities.